Bacterial infections are an increasing problem worldwide. The widespread use of conventional antibiotics has provoked the development of increasingly resistant bacterial strains along with a depleting arsenal of sufficiently effective antibiotics.
As a result, research towards a deeper understanding of the mechanisms involved in bacterial infections is increasing. Thus, it has been discovered that a wide variety of bacteria coordinate their behaviour through cell-to-cell communication mediated by small, diffusible signals. This phenomenon has been dubbed quorum sensing (QS) [W. C. Fuqua et al., J. Bacteriol., 1994, 176, p 269-275], and is prevalent among bacteria that form complex surface attached communities called biofilms. It is estimated by the National Health Institute of America that 80% of persistent bacterial infections involve biofilms. QS enables bacteria to keep track of their numbers, and is considered to afford them a mechanism for minimizing host response by delaying the production of virulence factors until sufficient bacteria have been amassed to overwhelm host-defence mechanisms. Blocking of QS (either completely or partly) reinstates proper action of the host-defence system which subsequently eliminates bacterial intruders. Therefore, inhibition of QS presents an alternative therapeutic approach to the traditional antibiotic-mediated bacterial killing or growth inhibition.
It has been shown that garlic extract blocks QS and promotes rapid clearing of pulmonary infections caused by the bacterial strain Pseudomonas aeruginosa [T. Bjarnsholt et al., Microbiology, 2005, 151, p. 3873-3880]. Pseudomonas aeruginosa forms biofilm communities and utilizes QS through the production of signal molecules such as N-acyl homoserine lactones (AHL) and quinolones PQS). Garlic extract is also a well known natural medicine product having antibacterial and cholesterol lowering effects, among others.
Ajoene [(E,Z)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide] has previously been shown to be an active ingredient in the antimicrobial garlic extracts along with allicin and other organosulfur derivatives. However, in a study on ajoene (the E:Z=1:4 isomer used), although a strong growth inhibitor of Gram positive bacteria, ajoene was shown to have a varied inhibitory effect on Gram negative bacteria, and no measurable growth inhibitory effect on P. aeruginosa [R. Naganawa et al., Applied and Environmental Microbiology, 62, 1996, p. 4238-4242].
Present processes for the manufacture of ajoene derivatives include the isolation of ajoene derivatives or precursors from garlic extract and various synthetic approaches. Isolation from garlic is highly tedious and inefficient as the amount of ajoene available pr. Kg raw garlic is minute i.e. in the range of 10 mg and HPLC separation from related compounds in garlic is a necessity. Some synthetic and semi-synthetic approaches to ajoene and various derivatives have been disclosed in the prior art. Eric Block and co-workers have disclosed a synthetic approach starting from an oxidation of diallyl disulphide or derivatives and subsequent heating of the resulting crude allicin or derivatives to form ajoene and derivatives [EP 185324 and Block et al., J. Am. Chem. Soc., 1986, 108, p. 7045-7055]. Apitz-Castro and co-workers have presented a semi-synthetic approach where allicin (allyl 2-propenethiosulfinate) is isolated from garlic bulbs and subsequently heated and treated with a lower alkyl alcohol to produce (E,Z)-ajoene derivatives [U.S. Pat. No. 4,665,088]. Recently a method of converting extracted allicin to ajoene using acetic acid in acetone was also reported, however crude impure starting material was used (i.e. crude allicin extracted from garlic) [WO 2010/100486]. However, the yields in the above methods have often been poor or purification by HPLC chromatography has been necessary leading to low yields of ajoene at high purification costs. Furthermore these synthetic approaches often lack control of the (E,Z)-isomeric outcome of the reaction, while still maintaining high yields.
Consequently, an alternative and improved method for the manufacture of ajoene derivatives would be advantageous.
It has now surprisingly been found that the synthesis of ajoene derivatives can be achieved in higher yields and better crude purities than previously obtained, while being able to vary the ratio of (E,Z) isomers in the final product via specific variations in the reaction conditions. Also, it was surprisingly found that certain mixtures of ajoene derivatives and/or isomers including those obtained by the above synthetic process and applying a single chromatographic purification step were effective QS inhibitors of biofilm forming bacteria, both when applied alone and in composition with conventional antibiotics.